The present invention is generally directed to metal chelating compositions and to methods for making and using the same for protein purification, detection or binding and, in particular, to nitrilotriacetic acid derivatives that have improved binding specificity and stability and to methods for making and using these nitrilotriacetic acid derivatives for protein purification, protein detection or protein binding.
Metal chelate affinity chromatography has been used as a technique for the purification of proteins for many years. Early resins used in this process were simple chelators such as iminodiacetic acid (IDA) coupled to agarose supports (Porath et al. Nature, 258:598-599, 1975) and charged with various metals such as Cu2+, Zn2+ and Ni2+. These resins were found to selectively capture proteins and peptides from natural sources (Porath and Olin, Biochemistry, 22:1621, 1983; Lonnerdal and Keen, J. Appl. Biochem., 4:203, 1983; Sulkowski, Protein Purification: Micro to Macro, pages 149-162, Edited by R. Burgess, Published by Liss New York, N.Y., 1987). With the advent of molecular biological techniques, metal chelate chromatography assumed a more important role in the purification of proteins with the use of a 6-histidine tag. See, for example, Dobeli et al., U.S. Pat. No. 5,284,933. The poly histidine tag bound very strongly to the immobilized nickel and could be used for the identification and purification of these recombinant molecules. The tridentate chelator IDA was quite selective for these tagged proteins but the nickel was found to leach slowly from the resin reducing the capacity and causing interference with some downstream uses of the proteins.
More recently, a tetradentate chelator known as nitrilotriacetic acid resin was developed for use with metals having six coordination sites. This resin has become the preferred resin for the purification of poly histidine containing proteins since it has very little metal leaching and good selectivity. However, considerable amount of effort is required to obtain this selectivity. For example, the addition of various amounts of imidazole is necessary to determine whether the resin will bind the protein selectively and the capacity of the resin for the protein must be optimized to achieve the desired results (Janknecht et all, Proc. Natl. Acad. Sci., 88:8972-8976, 1991, Schmitt et all., Molecular Biology Reports, 88:223-230, 1993).
In U.S. Pat. No. 4,877,830, Dobeli et al. describe nitrilotriacetic acid resins suitable for protein purification represented by the general formula:
[carrier matrix]-spacer-NHxe2x80x94(CH2)xxe2x80x94CH(COOH)xe2x80x94N(CH2COOxe2x80x94)2Ni2+
wherein x is 2, 3 or 4, the carrier matrix is one used in affinity or gel chromatography such as cross-linked dextrans, agarose or polyacrylamides, and the spacer is preferably xe2x80x94Oxe2x80x94CH2xe2x80x94CH(OH)xe2x80x94CH2xe2x80x94 or xe2x80x94Oxe2x80x94COxe2x80x94. Dobeli et al., U.S. Pat. No. 4,877,830 at col. 2, lines 23-37. These resins are prepared by reacting an N-terminal protected compound of the formula:
Rxe2x80x94HNxe2x80x94(CH2)xxe2x80x94CH(NH2)xe2x80x94COOH
wherein R is an amino protecting group and x is 2,3 or 4, with bromoacetic acid in an alkaline medium and subsequently cleaving off the protecting group and reacting this product with an activated resin. See, e.g., Hochuli et al., Journal of Chromatography, 411(1987) 177-184.
In U.S. Pat. No. 5,625,075, Srinivasan et al. describe a metal radionuclide chelating compound having multiple sulfur and nitrogen atoms. These chelating compounds incorporate two nitrogen atoms and three sulfur atoms, two nitrogen atoms and four sulfur atoms, or three nitrogen atoms and three sulfur atoms.
While these compounds provide improved specificity relative to some resins containing nitrilotriacetic acid derivatives, a need remains for chelating compounds having greater binding specificity for polyhistidine containing proteins.
Among the objects of the present invention is the provision of metal chelating compositions and to metal chelates which are relatively stable and provide superior binding specificity for protein or polypeptide purification, protein or polypeptide detection or protein or polypeptide binding, and the provision of processes for the preparation and use of such compositions.
Briefly, therefore, the present invention is directed to a metal chelating composition having the formula: 
wherein
Q is a carrier;
S1 is a spacer;
L is xe2x80x94Axe2x80x94Txe2x80x94CH(X)xe2x80x94 or xe2x80x94C(xe2x95x90O)xe2x80x94;
A is an ether, thioether, selenoether, or amide linkage;
T is a bond or substituted or unsubstituted alkyl or alkenyl;
X is xe2x80x94(CH2)kCH3, xe2x80x94(CH2)kCOOH, xe2x80x94(CH2)kSO3H, xe2x80x94(CH2)kPO3H2, xe2x80x94(CH2)kN(J)2, or xe2x80x94(CH2)kP(J)2;
k is an integer from 0 to 2;
J is hydrocarbyl or substituted hydrocarbyl;
Y is xe2x80x94COOH, xe2x80x94H, xe2x80x94SO3H, xe2x80x94PO3H2, xe2x80x94N(J)2, or xe2x80x94P(J)2;
Z is xe2x80x94COOH, xe2x80x94H, xe2x80x94SO3H, xe2x80x94PO3H2, xe2x80x94N(J)2, or xe2x80x94P(J)2; and
i is an integer from 0 to 4.
The present invention is further directed to a metal chelate comprising a metal and the metal chelating composition of the present invention.
The present invention is further directed to a process for the purification or detection of a polypeptide or other composition having an affinity for a metal chelate. The process comprising contacting the composition with a metal chelate, the metal chelate comprising a metal and the metal chelating composition of the present invention.
The present invention is further directed to a process for the preparation of a mono- or dicarboxylated amine. The process comprises combining an amine and an oxoacid in the presence of a reducing agent. The amine has the formula R2R3NH wherein R2 is hydrocarbyl or substituted hydrocarbyl and R3 is hydrogen, hydrocarbyl or substituted hydrocarbyl.
Other objects and features will be in part apparent and in part pointed out hereinafter.
The linkage between the chelator and the resin was found by us to be an important parameter for the selectivity of the resin for polyhistidine tagged proteins. Conventional nitrilotriacetic acid resin has a positively charged amine linkage that acts as a binding site for any negatively charged molecule which may interfere with the binding of the protein to the coordination sites offered by the immobilized metal. Oxygen, sulfur, selenium and amides have some affinity for metals which may provide enhanced chelation properties, binding the metal more firmly than traditional tetradentate chelators having positive amine linkages. In addition, the use of a non-charged atoms between the nitrilo nitrogen and the carrier appears to reduce non-specific binding of proteins.
The metal chelating compositions of the present invention are capable of forming relatively stable chelates with metal ions and, advantageously, the presence of ether (xe2x80x94Oxe2x80x94), thioether (xe2x80x94Sxe2x80x94), selenoether (xe2x80x94Sexe2x80x94) or amide ((xe2x80x94NR1(Cxe2x95x90O)xe2x80x94) or (xe2x80x94(Cxe2x95x90O)NR1xe2x80x94) wherein R1 is hydrogen or hydrocarbyl) linkages within the chelating composition contributes to the specificity of the resulting chelate when it is used for the separation or purification of molecules such as proteins, phosphoproteins, peptides, phosphopeptides, DNA, RNA, oligonucleotides, drugs, and synthetic and natural products that have an affinity for metal chelates such as clustered histidines or poly histidines.
In general, the chelating compositions of the present invention correspond to composition (1) shown in the structure below: 
wherein
Q is a carrier;
S1 is a spacer;
L is xe2x80x94Axe2x80x94Txe2x80x94CH(X)xe2x80x94 or xe2x80x94C(xe2x95x90O)xe2x80x94;
A is an ether, thioether, selenoether, or amide linkage;
T is a bond or substituted or unsubstituted alkyl or alkenyl;
X is xe2x80x94(CH2)kCH3, xe2x80x94(CH2)kCOOH, xe2x80x94(CH2)kSO3H, xe2x80x94(CH2)kPO3H2, xe2x80x94(CH2)kN(J)2, or xe2x80x94(CH2)kP(J)2, preferably xe2x80x94(CH2)kCOOH or xe2x80x94(CH2)kSO3H;
k is an integer from 0 to 2;
J is hydrocarbyl or substituted hydrocarbyl;
Y is xe2x80x94COOH, xe2x80x94H, xe2x80x94SO3H, xe2x80x94PO3H2, xe2x80x94N(J)2, or xe2x80x94P(J)2, preferably, xe2x80x94COOH;
Z is xe2x80x94COOH, xe2x80x94H, xe2x80x94SO3H, xe2x80x94PO3H2, xe2x80x94N(J)2, or xe2x80x94P(J)2, preferably, xe2x80x94COOH; and
i is an integer from 0 to 4, preferably 1 or 2.
In general, the carrier, Q, may comprise any solid or soluble material or compound capable of being derivatized for coupling. Solid (or insoluble) carriers may be selected from a group including agarose, cellulose, methacrylate co-polymers, polystyrene, polypropylene, paper, polyamide, polyacrylonitrile, polyvinylidene, polysulfone, nitrocellulose, polyester, polyethylene, silica, glass, latex, plastic, gold, iron oxide and polyacrylamide, but may be any insoluble or solid compound able to be derivatized to allow coupling of the remainder of the composition to the carrier, Q. A preferred solid carrier is agarose or a high-throughput screening microtiterplate. Soluble carriers include proteins, nucleic acids including DNA, RNA, and oligonucleotides, lipids, liposomes, synthetic soluble polymers, proteins, polyamino acids, albumin, antibodies, enzymes, streptavidin, peptides, hormones, chromogenic dyes, fluorescent dyes, flurochromes or any other detection molecule, drugs, small organic compounds, polysaccharides and any other soluble compound able to be derivatized for coupling the remainder of the composition to the carrier, Q. Proteins or polysaccharides are the preferred carrier.
The spacer, S1, which flanks the carrier comprises a chain of atoms which may be saturated or unsaturated, substituted or unsubstituted, linear or cyclic, or straight or branched. Typically, the chain of atoms defining the spacer, S1, will consist of no more than about 25 atoms; stated another way, the backbone of the spacer will consist of no more than about 25 atoms. More preferably, the chain of atoms defining the spacer, S1, will consist of no more than about 15 atoms, and still more preferably no more than about 12 atoms. The chain of atoms defining the spacer, S1, will typically be selected from the group consisting of carbon, oxygen, nitrogen, sulfur, selenium, silicon and phosphorous and preferably from the group consisting of carbon, oxygen, nitrogen, sulfur and selenium. In addition, the chain atoms may be substituted or unsubstituted with atoms other than hydrogen such as hydroxy, keto (xe2x95x90O), or acyl such as acetyl. Thus, the chain may optionally include one or more ether, thioether, selenoether, amide, or amine linkages between hydrocarbyl or substituted hydrocarbyl regions. Exemplary spacers, S1, include methylene, alkyleneoxy (xe2x80x94(CH2)aOxe2x80x94), alkylenethioether (xe2x80x94(CH2)aSxe2x80x94), alkyleneselenoether (xe2x80x94(CH2)aSexe2x80x94), alkyleneamide (xe2x80x94(CH2)aNR1(Cxe2x95x90O)xe2x80x94), alkylenecarbonyl (xe2x80x94(CH2)aCO)xe2x80x94, and combinations thereof wherein a is generally from 1 to about 20 and R1 is hydrogen or hydrocarbyl, preferably alkyl. In one embodiment, the spacer, S1, is a hydrophilic, neutral structure and does not contain any amine linkages or substituents or other linkages or substituents which could become electrically charged during the purification of a polypeptide.
As noted above, the linker, L, may be xe2x80x94Axe2x80x94Txe2x80x94CH(X)xe2x80x94 or xe2x80x94C(xe2x95x90O)xe2x80x94. When L is xe2x80x94Axe2x80x94Txe2x80x94CH(X)xe2x80x94, the chelating composition corresponds to the formula: 
wherein Q, S1, A, T, X, Y, and Z are as previously defined. In this embodiment, the ether (xe2x80x94Oxe2x80x94), thioether (xe2x80x94Sxe2x80x94), selenoether (xe2x80x94Sexe2x80x94) or amide ((xe2x80x94NR1(Cxe2x95x90O)xe2x80x94) or (xe2x80x94(Cxe2x95x90O)NR1xe2x80x94) wherein R1 is hydrogen or hydrocarbyl) linkage is separated from the chelating portion of the molecule by a substituted or unsubstituted alkyl or alkenyl region. If other than a bond, T is preferably substituted or unsubstituted C1 to C6 alkyl or substituted or unsubstituted C2 to C6 alkenyl. More preferably, A is xe2x80x94Sxe2x80x94, T is xe2x80x94(CH2)nxe2x80x94, and n is an integer from 0 to 6, typically 0 to 4, and more typically 0, 1 or 2.
When L is xe2x80x94C(xe2x95x90O)xe2x80x94, the chelating composition corresponds to the formula: 
wherein Q, S1, i, Y, and Z are as previously defined.
In a preferred embodiment of the present invention, the sequence xe2x80x94S1xe2x80x94Lxe2x80x94, in combination, is a chain of no more than about 35 atoms selected from the group consisting of carbon, oxygen, sulfur, selenium, nitrogen, silicon and phosphorous, more preferably only carbon, oxygen sulfur and nitrogen, and still more preferably only carbon, oxygen and sulfur. To reduce the prospects for non-specific binding, nitrogen, when present, is preferably in the form of an amide moiety. In addition, if the carbon chain atoms are substituted with anything other than hydrogen, they are preferably substituted with hydroxy or keto. In a preferred embodiment, L comprises a portion (sometimes referred to as a fragment or residue) derived from an amino acid such as cystine, homocystine, cysteine, homocysteine, aspartic acid, cysteic acid or an ester thereof such as the methyl or ethyl ester thereof.
Exemplary chelating compositions of the present invention include the following: 
wherein Q is a carrier and Ac is acetyl.
Advantages are gained by the use of neutral ether, thioether, selenoether or amide linkage(s) in the linking moiety instead of positively charged amine linkages. Oxygen, sulfur, and selenium atoms and amides have some affinity for metals which may provide enhanced chelation properties, binding the metal more firmly than traditional tetradentate chelators having positive amine linkages. In addition, the use of a non-charged atoms between the nitrilo nitrogen and the carrier appears to reduce non-specific binding of proteins. Use of S, O, Se or amide in the linking moiety, L, therefore, in place of amine or other charged moieties tends to increase the stability and specificity of the composition for protein purification.
In one embodiment, metal chelating compositions (1) of the present invention may be derived from compositions having the general formula: 
wherein A, T, X, Y, Z and i are as previously defined. Preferably, composition (2) is represented by one of the following formulae:
HSxe2x80x94(CH2)nxe2x80x94CH(CH2COOH)xe2x80x94N(CH2COOH)2
HSxe2x80x94(CH2)nxe2x80x94NHCOxe2x80x94CH(CH2SO3xe2x88x92)xe2x80x94N(CH2COOH)2
and
H2Nxe2x80x94(CH2)nxe2x80x94NHCOxe2x80x94CH(CH2SO3xe2x88x92)xe2x80x94N(CH2COOH)2
wherein n is 1 or 2.
Compositions corresponding to structure (2) in which at least one of X, Y and Z comprises a carboxylic acid moiety may be prepared by reductive alkylation of an amine. In general, mono- and dicarboxylated amines may be prepared by reacting an amine having the formula R2R3NH wherein R2 is hydrocarbyl or substituted hydrocarbyl and R3 is hydrogen, hydrocarbyl or substituted hydrocarbyl with an oxoacid, such as glyoxylic acid, in the presence of a reducing agent such as a pyridine-borane complex, dimethylborane, trimethylborane, sodium cyanoborohydride. When the amine is an amino acid such as cystine, homocystine, cysteine, homocysteine, aspartic acid, cysteic acid or an ester thereof such as the methyl or ethyl ester thereof, the reaction advantageously produces a nitrilotriacetic acid derivative. For example, a nitrilotriacetic acid derivative of cystine may be prepared by combining cystine, an oxoacid such as glyoxylic acid, and a mild reducing agent; alcohol may preferably be included to aid in the clarification of the solution. Alternatively, other methods known in the art may be used for the preparation of composition (2), including haloalkylacids.
Composition (2) may be immobilized to form composition (1) by covalently attaching a chemical spacer, S1, to the linker, L, by any method known in the art and then reacting the carrier with the spacer, S1, to form a carrier-spacer chelate complex of composition (1). In another embodiment, carrier Q is first reacted with the spacer, S1, to form a carrier-spacer complex. Thereafter, the carrier-spacer complex is attached to the chelate-complex through the linker, L, to form composition (1).
In some instances, it is advantageous to activate the carrier, Q, with S1 prior to the attachment of the chelating portion of the molecule. In these instances where Q is an agarose resin, it may be activated using epichlorohydrin, tetrabutyldiglycidyl ether or any substance capable of activating a carrier.
A metal chelate may be formed by addition of a metal or a metal oxide to chelating composition (1) or composition (2) of the present invention. For example, a metal chelate of the present invention (in immobilized form) is represented by the following formula:
Qxe2x80x94S1xe2x80x94Axe2x80x94Txe2x80x94CH[((CH2)kxe2x80x94X)xe2x80x94N((CH2)ixe2x80x94Y)xe2x80x94(CH2)ixe2x80x94Z]M
wherein Q, S1, A, i, J, k, T, X, Y, and Z are as defined above and M comprises any metal or metal oxide capable of forming a chelate. Preferred metals and metal oxides include Ni, Hg, Ga, Cu, Ru, Co, Cd, Mg, Mn, Ti, In, Zn, Tc, Rh, Pd, Re, Fe, Au, Pb, and Bi, with Fe, Cu, Co, Au, and Ni being preferred for most applications. In general, the metal, M, preferred for a given application is dependant upon the specific binding capabilities of the chelating portion of composition (1) or (2) and on the compound to be bound or purified. For example, when X, Y and Z are xe2x80x94COOH, M is optimally Ni for purifying proteins with poly histidine sequences. When the compound is a phophoprotein, a phosphopeptide or a phosphate containing molecule, M is optimally Fe or Ga.
Definitions
The xe2x80x9chydrocarbylxe2x80x9d moieties described herein are organic compounds or radicals consisting exclusively of the elements carbon and hydrogen. These moieties include alkyl, alkenyl, alkynyl, and aryl moieties. These moieties may be substituted or unsubstituted and preferably are substituted or unsubstituted alkyl. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbyl groups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwise specified, these moieties comprise 1 to 20 carbon atoms.
Unless otherwise specified, the alkyl groups described herein are preferably lower alkyl containing from one to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight, branched chain or cyclic and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals.
Unless otherwise specified, the alkenyl groups described herein are preferably lower alkenyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals.
Unless otherwise specified, the alkynyl groups described herein are preferably lower alkynyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like. They may be substituted with aliphatic or cyclic hydrocarbyl radicals.
Unless otherwise specified, the aryl moieties described herein contain from 6 to 20 carbon atoms and include phenyl. They may be hydrocarbyl substituted with the various substituents defined herein. Phenyl is the more preferred aryl.
The substituted hydrocarbyl moieties described herein are hydrocarbyl moieties which are substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom. These substituents are other than hydroxyl and include lower alkoxy such as methoxy, ethoxy, butoxy; halogen such as chloro or fluoro; ethers; acetals; ketals; esters; heteroaryl such as furyl or thienyl; alkanoxy; acyl; acyloxy; nitro; amino; and amido.
The acyl moieties described herein contain hydrocarbyl, substituted hydrocarbyl or heteroaryl moieties. They have the general formula xe2x80x94C(O)X wherein X may include hydrocarbyl, hydrocarbyloxy, hydrocarbylamino or hydrocarbylthio.
A protein, as used herein, includes antibodies, enzymes, hemoglobin, hormones, polypeptides and peptides; and may be an intact molecule, a fragment thereof, or a functional equivalent thereof; and may be genetically engineered.
An antibody, as used herein, includes both polyclonal and monoclonal antibodies; and may be an intact molecule, a fragment thereof; and may be genetically engineered.